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. 2020 Jul 6;29(15):2535–2550. doi: 10.1093/hmg/ddaa141

Figure 4.

Figure 4

The three novel identified enhancers co-operate to drive upregulated activity of the minimal ZEB2 promoter in a cell state/time specific manner. (A) schematic overview of combinatorial luciferase reporter-based constructs used in this study. (B) Luciferase assay performed in differentiating iPSCs transfected with a luciferase-based construct containing the three identified enhancers together with the minimal promoter of ZEB2 shows differential luciferase activation according the cell state A basal level of activation can be observed already at undifferentiated, D0, state. Similar level is also detected at mature NPC state, D15, while the highest expression is at D6, neural rosette/early NPC state. (C) Luciferase reporter assay of the whole panel of constructs, transiently transfected to heterologous HEK293T cells (see also the Discussion section). As for NPCs, the three enhancers positively co-operate. MP, minimal promoter of ZEB2. Error bars represent standard deviation of three independent biological replicates. Statistical significance was calculated with GraphPad Prism using a multiple comparison one-way Anova test. Asterisks represent P-values: *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.